Società Italiana per lo Studio della Fibrosi Cistica...Rowe SM et al. NEJM, 2005; 352: 1992-2001...

Giulio Cabrini

Laboratorio di Patologia Molecolare

Unità di Biologia Molecolare dei Tessuti

Dipartimento di Patologia e Diagnostica

Azienda Ospedaliera Universitaria di Verona

La pipeline della ricerca pre-clinica in Italia

Roma 8 maggio 2015

Sessione plenaria:

Panorama della ricerca sulla fibrosi cistica in Italia

Società Italiana per lo Studio

della Fibrosi Cistica

Dichiarazione per conflitto di interessi Giulio Cabrini- non riceve sponsorizzazioni da industria coinvolta nella fibrosi cistica -è coinvolto in progetti di ricerca pre-clinica sulla fibrosi cistica finanziati dalla Fondazione Italiana per la Ricerca sulla Fibrosi Cistica sulla infiammazione, correttori di CFTR e potenziatori di CFTR

Lista progetti:- FFC #1/2013 Mechanism of action of trimethylangelicin in rescuing F508del CFTR

functional expression - FFC #8/2014 Design and synthesis of improved analogs of TMA for personalized

treatment of cystic fibrosis- FFC #17/2014 TRPA1 channels as novel targets for anti-inflammatory therapies in

CF lung- FFC #19/2014 Mitochondrial Ca2+ dependent inflammosome activation

exacerbates the P.aeruginosa-driven inflammatory response- FFC #24/2014 The role of GBA2 in cystic fibrosis lung inflammation: from

molecular mechanism to therapeuthic strategies- FFC #28/2014 In vitro study of potential pro-fibrotic effect of everolimus in

different hyuman airway cell lines. Searching for new biomarkers to optimize MTOR-inhibitor immunosuppressive treatment of cystic fibrosis patients undergoing lung transplantation

260 Progetti di ricerca

360 Pubblicazioni scientifiche peer-reviewed

450 Presentazioni congressuali

170 Gruppi/Istituti di ricerca

550 Ricercatori coinvolti

250 Contratti o borse assegnate

anni 2002-2014

Fondazione Italiana per la Ricerca sulla Fibrosi Cistica:progetti di ricerca selezionati e finanziati

(cortesia della Fondazione Italiana per la Ricerca sulla Fibrosi Cistica)

modificato da Amaral MD, Kunzelmann K. Molecular targeting of CFTR as a therapeutic approach to cystic fibrosis. Trends Pharmacol Sci. 2007 Jul;28(7):334-41.

difetto del gene CF

difetto della proteina CFTR

difetto del trasporto di cloro

ridotto liquido superficiale

ostruzione bronchiale

infezione batterica

infiammazione

insufficienza respiratoria

Fibrosi cistica nel polmone:dal gene alla insufficienza respiratoria

modificato da Amaral MD, Kunzelmann K. Molecular targeting of CFTR as a therapeutic approach to cystic fibrosis. Trends Pharmacol Sci. 2007 Jul;28(7):334-41.

difetto del gene CF

difetto della proteina CFTR

difetto del trasporto di cloro

ridotto liquido superficiale

ostruzione bronchiale

infezione batterica

infiammazione

insufficienza respiratoria

Principali aree della ricerca pre-clinica

1. Strategie per correggere il trasporto di cloro

2. Terapie anti-microbiche innovative

3. Nuovi bersagli per l’infiammazione polmonare

da Boucher RC. Evidence for airway surface dehydration as the initiating event in CF airway disease. J Intern Med. 2007 Jan;261(1):5-16.

LIQUIDORESPIRATORIOSUPERFICIALE

EPITELIO BRONCHIALE

NORMALE FIBROSI CISTICA

LA DISIDRATAZIONE DEL LIQUIDO RESPIRATORIO SUPERFICIALEE’ L’EVENTO INIZIALE DELLA MALATTIA POLMONARENELLA FIBROSI CISTICA

Aggiornamento consigliato: Bell SC, De Boeck K, Amaral MD. New pharmacological approaches for cystic fibrosis: promises, progress, pitfalls. Pharmacol Ther. 2015;145:19-34


Classe III non si attiva la funzione di CFTResempio: G551D“POTENZIATORI” DI CFTRattivano/aumentano il trasporto di cloro

Classe IICFTR non è presente su membrana cellulare esempio: DF508“CORRETTORI” DI CFTRcorreggono il difetto di localizzazione intracellulare

Classe ICodone di stop prematuro CFTR mancante oppure troncataesempio: G542X, R1162XMOLECOLE “READ-THROUGH”mascherano i codoni di stop prematuripermettendo la sintesi della proteina

modificata da Rowe SM et al. NEJM, 2005; 352: 1992-2001

update 05/2015


Progetti finanziati (2012-2014) n. 18


- Borgatti M The read-through approach for the treatment of cystic fibrosis caused by premature termination codons - Casavola V Mechanism of action of trimethylangelicin in rescuing F508del CFTR functional expression - Galietta L Task force for CF 2014: drug discovery for correctors and potentiators of F508del-CFTR Development of novel strategies to correct the chloride transport defect in cystic fibrosis - Gambari R Design and synthesis of improved analogs of trimethylangelicin (TMA) for personalized treatment of cystic fibrosis. - Lentini L Identification and validation of novel molecules obtained by integrated computational and experimental approaches for the read-through of PTCs in CF cells. - Loi R An induced pluripotent stem cell (iPS)-based approach for the repopulation and phenotypic correction of cystic fibrosis airway epithelium


- Lucarelli M Study of the pathogenetic and therapeutic role of the Epithelial Na+ channel (ENaC) in CF and CF-like disease - Luini A A systems biology approach to the correction of Cystic Fibrosis: from building a network of proteostasis regulatory pathways to combinatorial targeting. - Mazzei M ΔF508-CFTR correctors deriving from computational design and from safe natural compounds for a prompt clinical application - Melotti P Testing CFTR in epithelial organoids for drug development and diagnosis of cystic fibrosis. - Messina G Vessel associated progenitor cells as a promising cell-based approach to treat cystic fibrosis disease - Moran O The molecular structure and the folding of the whole Cystic Fibrosis Transmembrane Conductance Regulator (CFTR): correctors sites.




- Pagani F An RNA based approach based on ExSpeU1 for correction of CFTR splicing defects: analysis of efficacy in primary bronchial cells. - Pedemonte N Modulation of post-translational modification and quality control system as a novel therapeutic strategy for Cystic Fibrosis - Rusnati M Development of novel methodologies for the identification of CFTR-targeted drugs: a multidisciplinary approach using Real Time Surface Plasmon Resonance interaction assay supported by bioinformatics strategies on HPC infrastructures. - Sorio C Establishment of a semi-automated evaluation of CFTR function in blood cells for clinical applications - Venerando A A kinase-directed approach to rescue functionality of F508del CFTR.



First HTS

Second HTS

Chemical Library

NMD+/- yeast model carrying PTCsin a dual-fluo reporter system

Human cellular model carrying PTCsin GFP reporter gene

Human cellular model carrying PTCs in CFTR gene

Read-through molecules

Optimal combination to reach maximal levels of CFTR protein and restoring of CFTR channel activity

NMD inhibitors or correctors/potentiators

Progetti FFC #1/2010 e #2/2012

Molecole read-through per le mutazioni da stop prematuro

modificato da Linde e Kerem (2008) Trend Genet 24: 552-563

Coordinatrice: Monica Borgatti – Università FerraraNicola Altamura – CNR BariAlberto Bresciani – IRBM PomeziaGiulio Cabrini - Verona

cortesia di Monica Borgatti

cortesia di Luis J.V. Galietta

Laboratory of Molecular PathologyUniversity Hospital of Verona, IT

SCREENING DI EFFETTO ANTI-INFIAMMATORIO DI PIANTE MEDICINALICoordinatore: R. Gambari – Ferrara Partner: G. Cabrini – Verona (2005-2009)

Aegle marmelos contiene ilprincipio attivo anti-infiammatorio5-metossi-psoralene(2009)


1

F. Dall’Acqua e A. ChilinDept of Pharmaceutical SciencesUniversity of Padova, IT

L’analogo di sintesi chimica 4,6,4’-trimetilangelicina (TMA) è più potente di 5-metossi-psoralene (2010)


1 2Aegle marmelos contiene ilprincipio attivo anti-infiammatorio5-metossi-psoralene(2009)

Am J Physiol (Cell Physiol) 272: C976-88 (1997)

8-metossi-psoralene è un “potenziatore” della funzione di trasporto ionico di CFTR

anche 4,6,4’-trimetilangelicina (TMA) è un “potenziatore” della funzione di

trasporto di cloro di CFTR (2011)

3

4,6,4’-trimetilangelicina (TMA) è “correttore” di CFTR DF508 in monostrati di cellule epiteliali respiratorie primarie polarizzate

da pazienti affetti da fibrosi cistica (2014)

Cortesia di Valeria Casavola e Collmodificato da Favia M, Mancini MT, Bezzerri V, Guerra L, Laselva O, Abbattiscianni AC, Debellis L, Reshkin SJ, Gambari R, Cabrini G, Casavola V. Trimethylangelicin promotes the functional rescue of mutant F508del CFTR protein in cystic fibrosis airway cells. Am J Physiol Lung Cell Mol Physiol. 2014;307:L48-61.

4

Quesito:sono possibili molecole con doppia azione di “correttori” e “potenziatori” di CFTR DF508 ?

AS Verkman e Coll- Hybrid potentiator-corrector agonist (Bioorg Med Chem Lett. 2010;20:87-91)- Cyanoquinolines (Mol Pharmacol. 2011;80:683-93 e J Med Chem. 2012;55:1242-51)

LJV Galietta e Coll- Aminoarylthiazoles (J Biol Chem. 2011;286:15215-26)

4,6,4’-trimetilangelicina (TMA) una molecola con doppia azione per CFTR DF508 (“correttore” e “potenziatore”)

TMA per la fibrosi cistica: prospettive future

1Progetto FFC #1/2013Coordinatrice: V Casavola Coll: G Cabrini & R Gambari Mechanism of action of trimethylangelicin in rescuing F508del CFTR functional expression

2Progetto FFC # 8/2014 Coordinatore: R Gambari Coll: A Chilin & G Cabrini Design and synthesis of improved analogs of trimethylangelicin (TMA) for personalized treatment of cystic fibrosis

3

da Villella VR, Esposito S, Bruscia EM, Maiuri MC, Raia V, Kroemer G, Maiuri L.Targeting the Intracellular Environment in Cystic Fibrosis: Restoring Autophagyas a Novel Strategy to Circumvent the CFTR Defect. Front Pharmacol. 2013 Jan 21;4:1

Le alterazioni della autofagia in CF come bersaglio di correzione della CFTR DF508 e della eccessiva infiammazione

L. Maiuri e V. RaiaMilano e Napoli

p62

p62

aggresome

sequestration

proteasome

overload

Vps15

Vps34Ambra1

Atg14 Beclin1

PtdIns(3)P

Autophagy

p62

Rab5

Recycling

impaired

Vps15Vps34

Ambra1 Atg14Beclin1

The CF environment

Ubiqui n

HDAC6DF508-CFTR

Nature Cell Biol 2010; Autophagy 2012; Cell Death Diff 2013; Autophagy 2014.

Autophagy

blockedTG2

INFLAMMATION

Restoring autophagy (cysteamine)

Autophagy restored

p62

ER

Golgi

DF508 rescued

F508del rescue

PtdIns(3)P

Rab11 Rab5

Rab7

Recycling

restored

p62

DF508 PM disposal REDUCED

Functional D508 STABILIZED at PM after rescue

Negative loop INTERRUPTED

(loss of CFTR function – defective autophagy – impaired CFTR trafficking)

Targeting CK2 (EGCG)

PM stability

INFLAMMATION

IN DF508/DF508CELLS, IN MOUSE AND IN PATIENTS

Luigi Maiuri

STRATEGY OF CFTR REPAIR

cortesia di Luigi Maiuri

da Stoltz DA, Meyerholz DK, Welsh MJ. Origins of cystic fibrosis lung disease. N Engl J Med. 2015 Jan 22;372(4):351-62.

IL POLMONE DI UN PAZIENTE AFFETTO DA FIBROSI CISTICA HA RIDOTTO TRASPORTO MUCOCILIARE CHE FAVORISCE L’INFEZIONE BATTERICA


PSEUDOMONAS AERUGINOSA EVADE LE RISPOSTE IMMUNITARIENEL POLMONE DEL PAZIENTE AFFETTO DA FIBROSI CISTICA

da Cohen TS, Prince A. Cystic fibrosis: a mucosal immunodeficiency syndrome. Nat Med. 2012 Apr 5;18(4):509-19.


update 05/2015




- Bergamini G Metalloproteases released by Pseudomonas aeruginosa clinical strains as virulent factors in CF: clinical correlations and chemical modulators- Bevivino A Investigating the airway microbiome in cystic fibrosis patients with a severe decline in lung function: an opportunity for a personalized microbiomebased therapy - Bragonzi A Cystic fibrosis modifier genes related to P. aeruginosa lung disease. - Briani F Exploring pyrazinamide derivatives as novel P. aeruginosa inhibitors: unexploited antibacterial molecules for a new antibiotics target - Cigana C Pathophysiological relevance of glycosaminoglycans in Pseudomonas aeruginosa chronic lung infections and validation of new therapeutic approaches to modulate inflammation and tissue remodeling - Cocuzza C Drug development of new beta-lactam and linezolid-like compounds active against Staphylococcus aureus isolated from cystic fibrosis patientes: in vitroand in vivo biological evaluation - Garlanda C Infections in cystic fibrosis patients: effect of PTX3 genetic variants on endogenous PTX3 production and function - Leoni L Anti-virulence therapy against Pseudomonas aeruginosa: identification of antibiofilm drugs and development of inhalable Niclosamide and Flucytosine formulations


- Mangoni ML Development and preclinical testing of a novel antimicrobial peptide to treat Pseudomonas aeruginosa-induced lung infections - Notomista E Inhalable dry powders for chemically-modified human Cationic AntiMicrobial Peptides (CAMPs): moving toward in vivo application - Pacello F Targeting extracellular Protein Disulphide Isomerase to control Burkholderia cenocepacia lung infections - Pini A Preclinical development of the antimicrobial peptide M33 and onset of regulatory procedures for clinical trials- Riccardi G A very promising drug against Burkholderia cenocepacia - Silipo A Naturally occurring antimicrobials to counteract lung infections in cystic fibrosis patients: Cecropin A-Melittin (CA-M) hybrid peptides and polymixins- Scocchi M Development of BMAP18 as a peptide drug in the lung bacterial infections: a study to improve its effectiveness in the CF pulmonary environment. - Tortoli E Transmissibility and clinical significance of Mycobacterium abscessus in patients with cystic fibrosis



Identificazione e validazione di nuovi antibiotici anti-Pseudomonas aeruginosa: l’esempio di POL7001Alessandra Bragonzi – Ospedale San Raffaele – Milano

& Polyphor, Allschwil, CH

cortesia di Alessandra Bragonzi

Progetto FFC # 10/2011

da Stoltz DA, Meyerholz DK, Welsh MJ. Origins of cystic fibrosis lung disease. N Engl J Med. 2015 Jan 22;372(4):351-62.

IL POLMONE DI UN PAZIENTE AFFETTO DA FIBROSI CISTICA È PRECOCEMENTE AFFETTO DA INFIAMMAZIONE

polmone di un paziente affetto da fibrosi cistica (età 3 mesi)con infiltrato di neutrofili (freccia) che ostruisce il lume bronchiale


I NEUTROFILI RILASCIANO PROTEASI E RADICALI DELL’OSSIGENOAMPLIFICANDO IL DANNO POLMONARE BATTERICO:L’INFIAMMAZIONE NEL POLMONE DEI PAZIENTI E’ RITENUTA “ECCESSIVA”


update 05/2015




- Battistoni A Role of high affinity zinc transporters in Pseudomonas aeruginosa ability to colonize the inflamed cystic fibrosis lung - Berlutti F Lactoferrin-loaded niosomes in reducing inflammation and infection of cystic fibrosis airways. - Cabrini G TRPA1 channels as novel molecular targets for anti-inflammatory therapies in CF lung - Corti A GSH inhalation therapies in CF: how useful, how safe? Set-up of a CF murine model for monitoring of inflammation in vivo and assessment of convenient alternatives. - Dechecchi MC Structure-activity relationships (SAR) of neoglycoconjugates derived from deoxynojirimycin as possible therapeutic agents for Cystic Fibrosis lung disease, by modulating the metabolism of sphingolipids CF - De Stefano Lipoic acid as a proteostasis regulator for the control of inflammation in cystic fibrosis




- Evangelista V Phosphodiesterases type-4 (PDE4) inhibitors and β 2-adrenergic agonists to reduce neutrophilic lung inflammation in cystic fibrosis. Preclinical studies and identification of biomarkers of efficacy - Fraziano M Preclinical study of a novel aerosol immunotherapeutic approach based on Janus-faced liposomes to enhance innate antimicrobial immunity - Hirsch E Targeting PI3Kγ scaffold function to activate airway CFTR, limit lung inflammation and promote bronchorelaxation in cystic fibrosis - Lleò MM Development of a CF, IL-8/NF-KB transgenic mouse model for the in vivolong-term monitoring of the inflammatory response induced by bacteria treated or not with azithromycin - Pilette C Impaired secretory IgA and mucosal immunity in cystic fibrosis: contribution to lung pathology and impaired defence against bacterial infection, and role of CFTR-related epithelial changes in the regulation of the receptor-mediated IgA transcytosis.




- Pinton P Mitochondrial Ca2+-dependent inflammasome activation exacerbates the P. aeruginosa-driven inflammatory response - Pizzo E Identification and characterization of LPS-neutralizing human peptides: potential tools to control inflammation in cystic fibrosis lung disease - Raia V The Heme-oxygenase 1 (HO-1) as modulator of Cystic Fibrosis lung disease - Recchiuti A Resolvin D1 for Targeting Chronic Lung Inflammation and Infection in Cystic Fibrosis - Romani L Targeting pathogenic pathways leading to inflammatory Th17 responses in cystic fibrosis: from drug discovery to preclinical validation. - Romano M Mechanisms and clinical implications of endothelial dysfunction in cystic fibrosis. - Signorelli P Sphingolipid targeting in inflammation and fungal infection




- Sonnino S The role of Glucocerebrosidase GBA2 in cystic fibrosis lung inflammation: from molecular mechanism to therapeutic strategies - Romani L Targeting pathogenic pathways leading to inflammatory Th17 responses in cystic fibrosis: a drug discovery approach - Romano M The role of vascular endothelium in cystic fibrosis inflammation - Strazzabosco M Cystic Fibrosis liver disease: the role of CFTR as regulator of epithelial innate immunity

LA CERAMIDE COME BERSAGLIO MOLECOLARE NELLA ECCESSIVA INFIAMMAZIONE POLMONARE

4

3

1accumuli di ceramide implicati nella iper-infiammazione polmonare

2

P.aeruginosa in bronchial cells

+ miglustat

2011

il miglustat riduce ceramide e riduce infiammazione

il bersaglio di miglustat è GBA2. (riducendo l'attività di GBA2 si riducono sia ceramide che iper-infiammazione)

Progetti FFC #16/2010 e #14/2012Coordinatrice: M.C. Dechecchi – VeronaS Sonnino – Milano & F Becq – Poitiers, F

nuovi bersagli e nuovi farmaci per ridurre la ceramide

cortesia di M. Cristina Dechecchi

http://ajrcmb.atsjournals.org/content/vol43/issue3/cover.dtl

Progetto FFC # 19/2014P. Pinton – FerraraG. Cabrini - Verona

TRPC TRPC

P. Aeruginosa

CFTR

2+Ca

2+Ca

2+Ca

2+Ca

2+Ca

2+Ca

2+Ca

2+Ca

Orai-1 Orai-1

flagellumflagellum

Type III ssType III ss

NUCLEUS NUCLEUS

ER ER

TM MT

IP R3 IP R3

VDACVDAC

VDACV AD C

MCU MCU

M

c

io

PAPs re ognit

n PP

ro

ni n

AMs ec

git o

IPAFIPAF

IPAFIPAF

NLRP3

NLRP3

NLRP3

NLRP3

NLRP3NLRP3

IL-1b processing IL-1b processing

Inflammatory responseInflammatory response

Pathological phenotypeNormal phenotype

ROS

ROS

cortesia di Alessandro Rimessi e Paolo Pinton

Rimessi A, Bezzerri V, Patergnani S, Marchi S, Cabrini G, Pinton P.Mitochondrial Ca2+-dependent NLRP3 activation exacerbates the Pseudomonas aeruginosa-driven inflammatory response in cystic fibrosis. Nature Communications 2015 Feb 4;6:6201

Normal phenotype Cystic fibrosis phenotype

da Bezzerri V, et al. Mapping the transcriptional machinery of the IL-8 gene in human bronchial epithelial cells. J Immunol. 2011 Dec 1;187(11):6069-81

BERSAGLIO FARMACOLOGICO ANTI-INFIAMMATORIO: I CANALI TRPA1 COME “AMPLIFICATORI” DELL’INFIAMMAZIONE

Progetto FFC #17/2014G Cabrini – VeronaR Nassini – FirenzeR Gambari - Ferrara

in breveLa ricerca pre-clinica sulla fibrosi cistica in Italia•si estende sulle principali aree della ricerca internazionale:

•richiederà nuove risorse e nuovi progetti per trasferire i risultati preclinici in studi clinici e in sviluppi industriali utili alla cura dei pazienti




ringraziamenti

M BorgattiA BragonziV CasavolaMC DechecchiLJV GaliettaR GambariL MaiuriV RaiaM Romano

per le slide cortesemente fornite

Laboratorio di Patologia Molecolare

Dipartimento di Patologia e Diagnostica

Azienda Ospedaliera Universitaria di Verona

M. Cristina DechecchiAnna Tamanini

Paola PrandiniLisa ProvezzaAlessandra SantangeloSilvia MunariPaolo MarchiSerena Domenichini

23 04 2015

ringraziamenti

per finanziamento sui progetti di ricerca

Società Italiana per lo Studio della Fibrosi Cistica...Rowe SM et al. NEJM, 2005; 352: 1992-2001...

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